The present invention relates to a process for producing neuraminidase and a method and a reagent, for the determination of a sialic acid-containing substance. More particularly, the present invention relates to a process for producing neuraminidase by culturing a microorganism belonging to the genus Micromonospora and a method and a reagent for the quantitative determination of a sialic acid-containing substance utilizing such neuraminidase.
Neuraminidase (EC 3.2.1.18) is a hydrolase which acts to liberate the sialic acid residue using, as a substrate, the ketoside having an .alpha.-configuration of a sialic acid (collective name of an acyl derivative of neuraminic acid) located at the terminal of sugar proteins, sugar lipids and the like which are important constituents of the living body.
It is known that neuraminidase is widely distributed in microorganisms such as virus, bacteria and actinomyces, and tissues of birds, mammals, etc. It is also known that neuraminidase can be used for the quantitative assay of sialic acid-containing substances in the living body such as serum.
Heretofore, neuraminidase has been obtained by culturing a neuraminidase producing microorganism in a culture vessel to which colominic acid, and a leaching liquid or extracts from various animal tissues are added as neuraminidase-inducing materials. [METABOLITE, 16 (5), 761 (1979), Biochim. Biophys. Acta, 350, 425-431 (1974), J. Biochem. 82, 1425-1433 (1977), J. Bacteriol. 119 (2), 394-400 (1974), Canadian J. Microbiology 18, 1007 (1972), Japanese Published Examined Patent Application No. 11991/75]. Such methods, however, suffer in that it is extremely difficult from an economical viewpoint to obtain and use these neuraminidase-inducing materials in large quantities. Accordingly, the known methods for producing neuraminidase are not economically feasible on a commercial scale and a process for industrially producing neuraminidase at low cost is in demand.
To this end, the present inventors have found that when microorganisms belonging to the genus Micromonospora are cultured in a culture medium, neuraminidase is produced in marked quantities in the culture liquor and can be readily recovered therefrom. It has also been found that the neuraminidase so produced is useful in a method and as a reagent for the quantitative determination of sialic acid-containing substances.